characterizing-rna-modifications-single-neurons-using-mass

Characterizing RNA Modifications in Single Neurons Using Mass Spectrometry

Post-transcriptional modifications (PTMs) of RNA represent an understudied mechanism involved in the regulation of translation in the central nervous ...

fluorescence-guided-matrix-assisted-laser-desorptionionization-with

Fluorescence-Guided Matrix-assisted Laser Desorption/Ionization with Laser-Induced Postionization Mass Spectrometry of Individual Rat Neural Cells

Single-cell measurements are critical to understanding the rich spatiochemical heterogeneity of the brain. Matrix-assisted laser/desorption ionization ...

University of Illinois Urbana-Champaign

Mass spectrometry imaging (MSI), a rapidly growing subfield of chemical imaging, employs mass spectrometry (MS) technologies to create single- and multi-dimensional localization maps for a variety of atoms and molecules. Complimentary to other imaging approaches, MSI provides high chemical specificity and broad analyte coverage. This powerful analytical toolset is capable of measuring the distribution of many classes of inorganics, metabolites, proteins, and pharmaceuticals in chemically and structurally complex biological specimens in vivo, in vitro, and in situ. The MSI approaches highlighted in this Methods in Molecular Biology volume provide flexibility of detection, characterization, and identification of multiple known and unknown analytes. The goal of this chapter is to introduce investigators who may be unfamiliar with MS to the basic principles of the mass spectrometric approaches as used in MSI. In addition to guidelines for choosing the most suitable MSI method for specific investigations, cross-references are provided to the chapters in this volume that describe the appropriate experimental protocols.

A mass spectrometry primer for mass spectrometry imaging.

Pinpointing a specific cell from within a relatively uniform cell population to determine its chemical content presents a challenging bioanalytical task. Immunocytochemistry is the classical method used to localize specific molecules and, hence, selected cells. Mass spectrometry also probes endogenous molecules such as neuropeptides within a cell. Here, these two approaches are hyphenated to allow microchemical analysis of immunocytochemical-selected peptidergic neurons. This two-step strategy utilizes antibody-based localization of cells containing selected biomarkers to isolate the cell(s) of interest, followed by peptidomic analysis via mass spectrometry. Applicable to a broad range of analyte and cell types, the strategy was used to successfully profile neuropeptides from individual immunostained insect neurons stored for up to 2 weeks as well as from tissues preserved for 42 weeks.

Targeted single-cell microchemical analysis: MS-based peptidomics of individual paraformaldehyde-fixed and immunolabeled neurons.

The metabolome refers to the entire set of small molecules, or metabolites, within a biological sample. These molecules are involved in many fundamental intracellular functions and reflect the cell's physiological condition. The ability to detect and identify metabolites and determine and monitor their amounts at the single cell level enables an exciting range of studies of biological variation and functional heterogeneity between cells, even within a presumably homogenous cell population. Significant progress has been made in the development and application of bioanalytical tools for single cell metabolomics based on mass spectrometry, microfluidics, and capillary separations. Remarkable improvements in the sensitivity, specificity, and throughput of these approaches enable investigation of multiple metabolites simultaneously in a range of individual cell samples.

Progress toward single cell metabolomics.

Understanding and controlling the interactions occurring between cells and engineered materials are central challenges toward progress in the development of biomedical devices. In this work, we describe materials for direct ink writing (DIW), an extrusion-based type of 3D printing, that embed a custom synthetic protein (RGD-PDL) within the microfilaments of 3D-hydrogel scaffolds to modify these interactions and differentially direct tissue-level organization of complex cell populations in vitro. The RGD-PDL is synthesized by modifying poly-d-lysine (PDL) to varying extents with peptides containing the integrin-binding motif Arg-Gly-Asp (RGD). Compositional gradients of the RGD-PDL presented by both patterned and thin-film poly(2-hydroxyethyl) methacrylate (pHEMA) substrates allow the patterning of cell-growth compliance in a grayscale form. The surface chemistry-dependent guidance of cell growth on the RGD-PDL-modified pHEMA materials is demonstrated using a model NIH-3T3 fibroblast cell line. The formation of a more complex cellular system-organotypic primary murine dorsal root ganglion (DRG)-in culture is also achieved on these scaffolds, where distinctive forms of cell growth and migration guidance are seen depending on their RGD-PDL content and topography. This experimental platform for the study of physicochemical factors on the formation and the reorganization of organotypic cultures offers useful capabilities for studies in tissue engineering, regenerative medicine, and diagnostics.

3D-Printed pHEMA Materials for Topographical and Biochemical Modulation of Dorsal Root Ganglion Cell Response.

Microfluidic and mass spectrometry (MS) methods are widely used to sample and probe the chemical composition of biological systems to elucidate chemical correlates of their healthy and disease states. Though matrix-assisted laser desorption/ionization-mass spectrometry (MALDI)-MS has been hyphenated to droplet microfluidics for offline analyses, the effects of parameters related to droplet generation, such as the type of oil phase used, have been understudied. To characterize these effects, five different oil phases were tested in droplet microfluidics for producing samples for MALDI-MS analysis. Picoliter to nanoliter aqueous droplets containing 0.1 to 100 mM γ-aminobutyric acid (GABA) and inorganic salts were generated inside a polydimethylsiloxane microfluidic chip and deposited onto a conductive glass slide. Optical microscopy, Raman spectroscopy, and MALDI-mass spectrometry imaging (MSI) of the droplet samples and surrounding areas revealed patterns of solvent and oil evaporation and analyte deposition. Optical microscopy detected the presence of salt crystals in 50-100 μm diameter dried droplets, and Raman and MSI were used to correlate GABA signals to the visible droplet footprints. MALDI-MS analyses revealed that droplets prepared in the presence of octanol oil led to the poorest detectability of GABA, whereas the oil phases containing FC-40 provided the best detectability; GABA signal was localized to the footprint of 65 pL droplets with a limit of detection of 23 amol. The effect of the surfactant perfluorooctanol on analyte detection was also investigated.

Droplet Microfluidics with MALDI-MS Detection: The Effects of Oil Phases in GABA Analysis.

Complex 3D organizations of materials represent ubiquitous structural motifs found in the most sophisticated forms of matter, the most notable of which are in life-sustaining hierarchical structures found in biology, but where simpler examples also exist as dense multilayered constructs in high-performance electronics. Each class of system evinces specific enabling forms of assembly to establish their functional organization at length scales not dissimilar to tissue-level constructs. This study describes materials and means of assembly that extend and join these disparate systems-schemes for the functional integration of soft and biological materials with synthetic 3D microscale, open frameworks that can leverage the most advanced forms of multilayer electronic technologies, including device-grade semiconductors such as monocrystalline silicon. Cellular migration behaviors, temporal dependencies of their growth, and contact guidance cues provided by the nonplanarity of these frameworks illustrate design criteria useful for their functional integration with living matter (e.g., NIH 3T3 fibroblast and primary rat dorsal root ganglion cell cultures).

Deterministic Integration of Biological and Soft Materials onto 3D Microscale Cellular Frameworks.

The pancreatic islets of Langerhans are clusters of cells that function as endocrine units synthesizing and releasing insulin and a range of additional peptide hormones. The structural and chemical characteristics of islets change during type 2 diabetes development. Although a range of metabolites including neurotransmitters has been reported in rodent islets, the involvement of these cell-to-cell signaling molecules within human pancreatic islets in the pathophysiology of type 2 diabetes is not well known, despite studies suggesting that these molecules impact intra- and inter-islet signaling pathways. We characterize the enigmatic cell-to-cell signaling molecules, d-serine (d-Ser) and d-aspartate (d-Asp), along with multiple classical neurotransmitters and related molecules, in healthy versus type 2 diabetes-affected human islets using capillary electrophoresis separations. Significantly reduced d-Ser percentage and gamma-aminobutyric acid (GABA) levels were found in type 2 diabetes-affected islets compared to healthy islets. In addition, the negative correlations of many of the signaling molecules, such as d-Ser percentage (r = -0.35), d-Asp (r = -0.32), serotonin (r = -0.42), and GABA (r = -0.39) levels, with hemoglobin A1c (HbA1c) levels and thus with the progression of type 2 diabetes further demonstrate the disruption in intra- or inter-islet signaling pathways and suggest that these cell-to-cell signaling molecules may be potential therapeutic targets.

d-Amino Acids and Classical Neurotransmitters in Healthy and Type 2 Diabetes-Affected Human Pancreatic Islets of Langerhans.

Approaches for the characterization of proteins/peptides in single cells of formaldehyde-fixed (FF) tissues via mass spectrometry (MS) are still under development. The lack of a general method for selectively eliminating formaldehyde-induced crosslinking is a major challenge. A workflow is shown for the high-throughput peptide profiling of single cells isolated from FF tissues, here the rodent pancreas, which possesses multiple peptide hormones from the islets of Langerhans. The heat treatment is enhanced by a collagen-selective multistep thermal process assisting efficient isolation of islets from the FF pancreas and, subsequently, their dissociation into single islet cells. Hydroxylamine-based chemical decrosslinking helped restore intact peptide signals from individual isolated cells. Subsequently, an acetone/glycerol-assisted cell dispersion was optimized for spatially resolved cell deposition onto glass slides, while a glycerol solution maintained the hydrated state of the cells. This sample preparation procedure allowed peptide profiling in FF single cells by fluorescence-guided matrix-assisted laser desorption ionization MS. Here, 2594 single islet cells were analyzed and 28 peptides were detected, including insulin C-peptides and glucagon. T-distributed stochastic neighbor embedding (t-SNE) data visualization demonstrated that cells cluster based on cell-specific pancreatic peptide hormones. This workflow expands the sample availability for single-cell MS characterization to a wide range of formaldehyde-treated tissue specimens stored in biobanks.

Chemical Decrosslinking-Based Peptide Characterization of Formaldehyde-Fixed Rat Pancreas Using Fluorescence-Guided Single-Cell Mass Spectrometry.

Elucidating the spatial-biochemical organization of the brain across different scales produces invaluable insight into the molecular intricacy of the brain. While mass spectrometry imaging (MSI) provides spatial localization of compounds, comprehensive chemical profiling at a brain-wide scale in three dimensions by MSI with single-cell resolution has not been achieved. We demonstrate complementary brain-wide and single-cell biochemical mapping via MEISTER, an integrative experimental and computational mass spectrometry framework. MEISTER integrates a deep-learning-based reconstruction that accelerates high-mass-resolving MS by 15-fold, multimodal registration creating 3D molecular distributions, and a data integration method fitting cell-specific mass spectra to 3D data sets. We imaged detailed lipid profiles in tissues with data sets containing millions of pixels, and in large single-cell populations acquired from the rat brain. We identified region-specific lipid contents, and cell-specific localizations of lipids depending on both cell subpopulations and anatomical origins of the cells. Our workflow establishes a blueprint for future developments of multiscale technologies for biochemical characterization of the brain.

Integrative Multiscale Biochemical Mapping of the Brain via Deep-Learning-Enhanced High-Throughput Mass Spectrometry.

Microbiome-derived metabolites are important for the microbiome-gut-brain axis and the discovery of new disease treatments. D-Alanine (D-Ala) is found in many animals as a potential co-agonist of the N-methyl-D-aspartate receptors (NMDAR), receptors widely used in the nervous and endocrine systems. The gut microbiome, diet and putative endogenous synthesis are the potential sources of D-Ala in animals, although there is no direct evidence to show the distribution and racemization of gut-absorbed L-/D-Ala with regards to host-microbe interactions in mammals. In this work, we utilized germ-free mice to control the interference from microbiota and isotopically labeled L-/D-Ala to track their biodistribution and racemization in vivo. Results showed time-dependent biodistribution of gut-absorbed D-Ala, particularly accumulation of gut-absorbed D-Ala in pancreatic tissues, brain, and pituitary. No endogenous synthesis of D-Ala via racemization was observed in germ-free mice. The sources of D-Ala in mice were revealed as microbiota and diet, but not endogenous racemization. This work indicates the importance of further investigating the in vivo biological functions of gut-microbiome derived D-Ala, particularly on NMDAR-related activities, for D-Ala as a potential signaling molecules in the microbiome-gut-brain axis.

Biodistribution and racemization of gut-absorbed L/D-alanine in germ-free mice.

The cell-to-cell signaling role of d-amino acids (d-AAs) in the mammalian endocrine system, particularly in the islets of Langerhans, has drawn growing interest for their potential involvement in modulating glucose metabolism. Previous studies found colocalization of serine racemase [produces d-serine (d-Ser)] and d-alanine (d-Ala) within insulin-secreting beta cells and d-aspartate (d-Asp) within glucagon-secreting alpha cells. Expressed in the islets, functional -methyl-d-aspartate receptors are involved in the modulation of glucose-stimulated insulin secretion and have binding sites for several d-AAs. However, knowledge of the regulation of d-AA levels in the islets during glucose stimulation as well as the response of islets to different levels of extracellular d-AAs is limited. In this study, we determined the intracellular and extracellular levels of d-Ser, d-Ala, and d-Asp in cultures of isolated rodent islets exposed to different levels of extracellular glucose. We found that the intracellular levels of the enantiomers demonstrated large variability and, in general, were not affected by extracellular glucose levels. However, significantly lower levels of extracellular d-Ser and d-Ala were observed in the islet media supplemented with 20 mM concentration of glucose compared to the control condition utilizing 3 mM glucose. Glucose-induced oscillations of intracellular free calcium concentration ([Ca]), a proxy for insulin secretion, were modulated by the exogenous application of d-Ser and d-Ala but not by their l-stereoisomers. Our results provide new insights into the roles of d-AAs in the biochemistry and function of pancreatic islets.

Relations between Glucose and d-Amino Acids in the Modulation of Biochemical and Functional Properties of Rodent Islets of Langerhans.

Spatial omics technologies can reveal the molecular intricacy of the brain. While mass spectrometry imaging (MSI) provides spatial localization of compounds, comprehensive biochemical profiling at a brain-wide scale in three dimensions by MSI with single-cell resolution has not been achieved. We demonstrate complementary brain-wide and single-cell biochemical mapping using MEISTER, an integrative experimental and computational mass spectrometry (MS) framework. Our framework integrates a deep-learning-based reconstruction that accelerates high-mass-resolving MS by 15-fold, multimodal registration creating three-dimensional (3D) molecular distributions and a data integration method fitting cell-specific mass spectra to 3D datasets. We imaged detailed lipid profiles in tissues with millions of pixels and in large single-cell populations acquired from the rat brain. We identified region-specific lipid contents and cell-specific localizations of lipids depending on both cell subpopulations and anatomical origins of the cells. Our workflow establishes a blueprint for future development of multiscale technologies for biochemical characterization of the brain.

Multiscale biochemical mapping of the brain through deep-learning-enhanced high-throughput mass spectrometry.

Achieving high-resolution and high signal-to-noise ratio (SNR) in vivo metabolic imaging via fast magnetic resonance spectroscopic imaging (MRSI) has been a longstanding challenge. This study combines the methods of relaxation enhancement (RE) and subspace imaging for the first time, enabling high-resolution and high-SNR in vivo MRSI of rodent brains at 9.4 T. Specifically, an RE-based chemical shift imaging sequence, which combines a frequency-selective pulse to excite only the metabolite frequencies with minimum perturbation of the water spins and a pair of adiabatic pulses to spatially localize the slice of interest, is designed and evaluated in vivo. This strategy effectively shortens the apparent T of metabolites, thereby increasing the SNR during relatively short repetition time ((T) compared with acquisitions with only spatially selective wideband excitations, and does not require water suppression. The SNR was further enhanced via a state-of-the-art subspace reconstruction method. A novel subspace learning strategy tailored for 9.4 T and RE acquisitions is developed. In vivo, high-resolution (e.g., voxel size of 0.6 × 0.6 × 1.5 mm) MRSI of both healthy mouse brains and a glioma-bearing mouse brain in 12.5 min has been demonstrated.

High-resolution H-MRSI at 9.4 T by integrating relaxation enhancement and subspace imaging.

Droplet microfluidics with push-pull and microdialysis sampling from brain slices, cultured cells and engineered tissues produce low volume mass limited samples containing analytes sampled from the extracellular space. This sampling approach coupled to mass spectrometry (MS) detection allows evaluation of time-dependent chemical changes. Our goal is an approach for continuous sampling and segregation of extracellular samples into picoliter droplets followed by the characterization of the droplets using nanoelectrospray ionization (nESI) MS. The main focus here is the optimization of the carrier oil for the microfluidic device that neither affects the stability of picoliter droplets nor compatibility with MS detection of a range of analytes.

1-Octanol-assisted ultra-small volume droplet microfluidics with nanoelectrospray ionization mass spectrometry.

Organoids are multicellular structures formed  from populations of individual cells allowing modeling of structural and functional aspects of organs and tissues in normal and diseased states. They offer unique opportunities to model and treat disease. Using a mouse embryonic stem cell line, we have cultured organoids that express markers of spinal cord motor neurons as well as motor neurons found within the peripheral nervous system. The morphology and select neurotransmitter content of the organoids and spinal cord explants were compared at different developmental time points. We found indications of maturation in the organoids over time, mirrored by similar trends in the spinal cord explants. Although the organoids contained the same neurotransmitters as the spinal cord explants, the developmental changes of these neurotransmitter levels were less marked in organoids. Given these differences, further work is required to optimize organoid growth conditions to better reproduce  models when using organoids to study development.

ArticleTotal : 2	Year
Publication title Cited by 3	2022
Publication title	2025

Article	Year
A mass spectrometry primer for mass spectrometry imaging. Methods in molecular biology (Clifton, N.J.)\| PubMed ID: 20680583	2010
Targeted single-cell microchemical analysis: MS-based peptidomics of individual paraformaldehyde-fixed and immunolabeled neurons. Chemistry & biology\| PubMed ID: 22921068	2012
Progress toward single cell metabolomics. Current opinion in biotechnology\| PubMed ID: 23246232	2013
3D-Printed pHEMA Materials for Topographical and Biochemical Modulation of Dorsal Root Ganglion Cell Response. ACS applied materials & interfaces\| PubMed ID: 28813592	2017
Droplet Microfluidics with MALDI-MS Detection: The Effects of Oil Phases in GABA Analysis. ACS measurement science Au\| PubMed ID: 34939077	2021
Deterministic Integration of Biological and Soft Materials onto 3D Microscale Cellular Frameworks. Advanced biosystems\| PubMed ID: 29552634	2017
d-Amino Acids and Classical Neurotransmitters in Healthy and Type 2 Diabetes-Affected Human Pancreatic Islets of Langerhans. Metabolites\| PubMed ID: 36144204	2022
Chemical Decrosslinking-Based Peptide Characterization of Formaldehyde-Fixed Rat Pancreas Using Fluorescence-Guided Single-Cell Mass Spectrometry. Analytical chemistry\| PubMed ID: 37040477	2023
Integrative Multiscale Biochemical Mapping of the Brain via Deep-Learning-Enhanced High-Throughput Mass Spectrometry. bioRxiv : the preprint server for biology\| PubMed ID: 37398021	2023
Biodistribution and racemization of gut-absorbed L/D-alanine in germ-free mice. Communications biology\| PubMed ID: 37587187	2023
Relations between Glucose and d-Amino Acids in the Modulation of Biochemical and Functional Properties of Rodent Islets of Langerhans. ACS omega\| PubMed ID: 38144114	2023
Multiscale biochemical mapping of the brain through deep-learning-enhanced high-throughput mass spectrometry. Nature methods\| PubMed ID: 38366241	2024
High-resolution H-MRSI at 9.4 T by integrating relaxation enhancement and subspace imaging. NMR in biomedicine\| PubMed ID: 38715469	2024
1-Octanol-assisted ultra-small volume droplet microfluidics with nanoelectrospray ionization mass spectrometry. Analytica chimica acta\| PubMed ID: 39155094	2024
Neurochemical and Morphological Comparisons of Motor Nerve Organoids and Spinal-Cord Explants. ACS chemical neuroscience\| PubMed ID: 39692746	2025

Stanislav S. Rubakhin

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